The present invention discloses the isolation of a key portion of the catalytic kinase region of vascular endothelial growth factor receptor 2 or VEGFR-2 through cloning, sequencing and x-ray crystallography. Also disclosed is the deletion of various amino acid residues from an area of the catalytic region called the kinase insert domain (KID). The resulting polypeptide retains comparable in vitro kinase activity to that of the wild-type KID and is not necessary for the catalytic activity of the polypeptide, and more importantly, allows complete crystallization of the protein such that it may be characterized by X-ray crystallography. The present invention further discloses x-ray crystallography data useful for identification and construction of therapeutic compounds in the treatment of various disease conditions associated with VEGFR-2.
Many physiological events including embryogenesis, organ development, estrus, and wound healing require vascular growth and remodeling (Folkman et al., (1992) J. Biol. Chem. 287, 10931-10934; Risau, W. (1995) FASEB J. 9, 926-933.). In addition to these beneficial processes, angiogenesis is also involved in the proliferation of disease states such as tumor growth, metastasis, psoriasis, rheumatoid arthritis, macular degeneration and retinopathy (Pepper, M. S., (1996) Vasc. Med 1, 259-266; Kuiper et al., (1998) Pharmacol. Res. 37, 1-16, 1998; Kumar and Fidler, (1998) In Vivo 18, 27-34; Szekanecz et al., (1998) J. Investig. Med. 45, 27-41; Tolentino and Adamis, (1988) Int. Ophthalmol. Clin. 38, 77-94. Of the signaling pathways known to influence vascular formation, these involving vascular endothelial growth factor (VEGF) haves been shown to be essential and selective for vascular endothelial cells (Dvorak et al., (1995) Am. J. Path. 146, 1029-1039; Thomas, K., (1996) J. Biol. Chem. 271, 603-606; Ferrara N. and Davis-Smyth, (1997) Endocrine Rev. 18, 4-25). The therapeutic potential of inhibiting the VEGF pathway has been directly demonstrated by anti-VEGF monoclonal antibodies which were active against a variety of human tumors (Borgstrxc3x6m et al, (1996) Cancer Res. 58, 4032-4039) and ischemic retinal disease (Adamis et al., (1996) Arch. Ophthalmol. 114, 66-71).
Normal vasculogenesis and angiogenesis play important roles in a variety of physiological processes such as embryonic development, wound healing, organ regeneration and female reproductive processes such as follicle development in the corpus luteum during ovulation and placental growth after pregnancy (Folkman and Shing, 1992). Uncontrolled vasculogenesis and/or angiogenesis has been associated with diseases, such as diabetes, as well as malignant solid tumors that rely on vascularization for growth. Klagsburn and Soker, (1993) Current Biology 3(10):699-702; Folkham, (1991) J. Natl., Cancer Inst. 82:4-6; Weidner, et al., (1991) New Engl. J. Med. 324:1-5.
Several polypeptides with in vitro endothelial cell growth promoting activity have been identified. Examples include acidic and basic fibroblastic growth factor (FGF), vascular endothelial growth factor (VEGF) and placental growth factor. Unlike FGF, VEGF has recently been reported to be an endothelial cell specific mitogen (Ferrara and Henzel, (1989) Biochem. Biophys. Res. Comm. 161:851-858; Vaisman et al., (1990) J. Biol. Chem. 265:19461-19566).
Thus, identification of the specific receptors to which VEGF binds is important to understanding of the regulation of endothelial cell proliferation. Two structurally related tyrosine kinases have been identified to bind VEGF with high affinity: the fit-1 receptor (Shibuya et al., (1990) Oncogene 5:519-524; De Vries et al., (1992) Science 255:989-991) and the KDR/FLK-1 receptor, discussed herein. Consequently, it had been surmised that RTKs may have a role in the modulation and regulation of endothelial cell proliferation.
Recent disclosures, such as information set forth in U.S. patent application Ser. Nos. 08/193,829, 08/038,596 and 07/975,750, strongly suggest that VEGF is not only responsible for endothelial cell proliferation, but also is the prime regulator of normal and pathological angiogenesis. See generally, Klagsburn and Soker, (1993) Current Biology3:699-702; Houck, et al., (1992) J. Biol. Chem. 267:26031-26037.
VEGF is a homodimeric cytokine that is expressed in at least four splice-variant forms of 121-206 residues (Ferrara and Davis-Smyth, 1997). Vascular endothelial cells express at least two high-affinity receptors for VEGF: VEGF-R1/Flt-1 and VEGFR-2/KDR. VEGF-R1 and VEGFR-2 are receptor tyrosine kinases each comprised of an extracellular domain that contains 7 immunoglobulin-like segments and binds VEGF, a short membrane spanning region, and a cytosolic domain possessing tyrosine kinase activity. The kinase domain directly follows the extracellular and juxtamembrane regions and is itself followed by another domain (post-kinase domain), which may function in binding of other proteins for signal transduction. These two receptors appear to have different signaling pathways and functions with VEGFR-2 being of primary importance in mitosis of endothelial cells (Waltenberger et al., (1994) J. Biol. Chem. 269, 26988-26995; Seetharm et al., (1995) Oncogene 10, 135-147; Shalaby et al., (1995) Nature 376, 576-579).
Both FGF and VEGF are potent angiogenic factors which induce formation of new capillary blood vessels. Transfection of human breast carcinoma cell line MCF-7 with FGF resulted in cell lines that form progressively growing and metastatic tumors when injected (s.c.) into nude mice. FGF may play a critical role in progression of breast tumors to an estrogen-independent, anti-estrogen resistant metastatic phenotype (McLeskey et al., (1993) Cancer Res. 53: 2168-2177). Breast tumor cells exhibited increased neovascularization, increased spontaneous metastasis and more rapid growth in vivo than did the non-transfected tumors. FGF has been shown to be
transforming in NIH-3T3 cells and implicated in tumorigenesis and metastasis of mouse mammary tumors. FGF overexpression conferred a tumorigenic phenotype on a human adrenal carcinoma cell line suggesting that FGF""s may also play a role in the transformation of epithelial cells. Polyclonal neutralizing antibodies to FGF inhibited tumor growth in Balb/c nude mice transplanted with K1000 cells (transfected with the leader sequence of bFGF) which form tumors in these mice (Hori et al., (1991) Cancer Res. 51: 6180-9184).
Due to the role of FGF in neovascularization, tumorigenesis and metastasis, there is a need in the art for FGF inhibitors as potent anti-cancer agents that exert their anti-FGF activity by preventing intracellular signaling of FGF.
VEGF, by contrast, is an endothelial cell-specific mitogen and an angiogenesis inducer that is released by a variety of tumor cells and expressed in human tumor cells in situ. Unlike FGF, transfection of cell lines with a cDNA sequence encoding VEGF, did not promote transformation, but did facilitate tumor growth in vivo (Ferrara, N., and Davis-Smyth, T. (1997)). Furthermore, administration of a polyclonal antibody which neutralized VEGF also inhibited growth of human rhabdomyosarcoma, glioblastoma multiforme and leiomyosarcoma cell lines in nude mice (Kim et al., (1993) Nature 362: 841-843).
In view of the importance of receptor tyrosine kinases (RTKs) to the control, regulation and modulation of endothelial cell proliferation and potentially vasculogenesis and/or angiogenesis, many attempts have been made to identify RTK xe2x80x9cinhibitorsxe2x80x9d using a variety of approaches, including the use of mutant ligands (U.S. Pat. No. 4,966,849), soluble receptors and antibodies (Application No. WO 94/10202; Kendall and Thomas, (1994) Proc. Natl. Acad. Sci. 90:10705-09; Kim, et al., 1993), RNA ligands (Jellinek, et al., (1994) Biochemistry 3:10450-56), protein kinase C inhibitors (Schuchter, et al., (1991) Cancer Res. 51:682-687); Takano, et al., (1993) Mol. Bio. Cell 4:358A; Kinsella, et al., (1992) Exp. Cell Res. 199:56-62; Wright, et al., (1992) J. Cellular Phys. 152:448-57) and tyrosine kinase inhibitors (WO 94/03427; WO 92/21660; WO 91/15495; WO 94/14808; U.S. Pat. No. 5,330,992; Mariani, et al., (1994) Proc. Am. Assoc. Cancer Res. 35:2268).
More recently, attempts have been made to identify small molecules which act as tyrosine kinase inhibitors. For example, bis monocyclic, bicyclic or heterocyclic aryl compounds (PCT WO 92/20642), vinyleng-azaindole derivatives (PCT WO 94/14808) and 1-cycloproppyl-4-pyridyl-quinolones (U.S. Pat. No. 5,330,992) have been described generally as tyrosine kinase inhibitors. Styryl compounds (U.S. Pat. No. 5,217,999), styryl-substituted pyridyl compounds (U.S. Pat. No. 5,302,606), certain quinazoline derivatives (EP Application No. 0 566 266 Al), selenoindoles and selenides (PCT WO 94/03427), tricyclic polyhydroxylic compounds (PCT WO 92/21660) and benzylphosphonic acid compounds (PCT WO 91/15495) have been described as compounds for use as tyrosine kinase inhibitors for use in the treatment of cancer. None of these compounds, however, have been previously associated
with the enzymatic function of the VEGFR-2 receptor. Likewise, none of these compounds have been associated with regulation of vasculogenesis and/or angiogenesis.
Therefore, there is a need in the art to develop small molecule antagonists of the PDGF, FGF, EGF and VEGF pathways individually or as a group. Moreover, if these cytokines signal through a common second messenger pathway within the cell, such antagonists will have broad therapeutic activity to treat or prevent the progression of a broad array of diseases, such as coronary restenosis, tumor-associated angiogenesis, atherosclerosis, autoimmune diseases, acute inflammation, certain kidney diseases associated with proliferation of glomerular or mesangial cells,
and ocular diseases associated with retinal vessel proliferation. The present invention was made by discovering a common signaling mechanism, a group of active therapeutic agents, shown to be active by a large number of and variety of predictive assays, and discovering a common intracellular signaling intermediate.
Based on sequence homology and overall domain structure, VEGFRs belong to the platelet-derived growth factor receptor family (PDGFR) which also includes PDGFRxcex1, PDGFRxcex2, the stem cell growth factor receptor (c-kit), and the colony stimulating factor-1 receptor (CSF-1R/c-fms) (van der Geer et al., (1994) Ann. Rev. Cell Biol. 10, 251-337). Compared to other protein kinases, members of this family contain an insert of approximately 65-97 residues, termed the kinase insert domain (KID), within the catalytic kinase domain relative to other protein kinases. Within the PDGFR family the KIDs are of varying length and low sequence homology. Deletion or mutation of the KID from PDGFRxcex1, PDGFRxcex2, c-kit, and CSF-1R have indicated that this domain is not necessary for intrinsic kinase activity but that it is important for the binding of other proteins involved in signal transduction, via autophosphorylation of KID tyrosine residues (Taylor et al., (1989) EMBO J. 8, 2029-2037; Heidaran et al., (1991) Mol. Cell. Biol. 11, 134-142; Yu etal., (1991) Mol. Cell. Biol. 11, 3780-3785; Kazlauskasetal., (1992) Mol. Cell. Biol. 12, 2534-2544; Lev et al., (1992) Proc. Natl. Acad. Sci. USA 89, 678-682; Reedjik etal., (1992) EMBO J. 11, 1365-1372; Bazenet et al., (1996) Mol. Cell. Biol. 16, 6926-6936). Although the signaling pathways and the specific role of the KID are still not fully determined for VEGFRs, the VEGFR-2 KID does contain two tyrosines which are known to be autophosphorylation sites (Dougher-Vermazen et al., (1994) Biochem. Biophys. Res. Comm. 205, 728-738).
Since the determination of the first cyclic AMP-dependent protein kinase (cAPK) structure (Knighton et al., (1991) Science 253, 407-413) a variety of protein kinase structures have been reported (reviewed in Johnson et al., (1996) Cell 85, 149-158). Among the receptor protein tyrosine kinases (RTKs), structures of the kinase domain of the insulin receptor (IRK) (Hubbard, et al., (1994) Nature 372, 746-754; Hubbard, (1997) EMBO J. 16, 5572-5581) and the fibroblast growth factor receptor-1 (FGFR1) (Mohammadi et al., (1996) Cell 86, 577-87; Mohammadi et al., (1997) Science 276, 955-960) have been determined.
The present invention discloses the generation, kinetic characterization, and structure determination of a modified kinase domain of the VEGFR-2 protein, containing 18 residues of the 68 residue KID. This 2.4 xc3x85 crystal structure of the phosphorylated VEGFR-2 catalytic domain is the first reported structure of a kinase domain of the PDGFR family. This structure provides insights into the orientation of the KID domain of VEGFR-2 which may be relevant to other PDGFR family members. Furthermore, as inhibition of VEGFR-2 kinase has broad clinical applications, this structure provides a three-dimensional description of the target for structure-based design of small molecule VEGFR-2 inhibitors as therapeutic agents.
It is an object of the present invention to disclose an effective method for screening candidate compounds that are specifically agonists or antagonists of various proteins which can be included in the receptor tyrosine kinase family (RTK) by crystallizing RTKs and particularly the VEGFR-2 receptor in order to use molecular modeling of the x-ray crystallography data to model the binding of candidate compounds.
There is disclosed a method for designing and screening potentially therapeutic compounds with activities such as: (1) inhibiting new blood vessel formation that is useful for treating or preventing progression of diabetic retinopathy, cavernous hemangiomas, Kaposi""s sarcoma, tumors composed of endothelial-like cells, and growth of cancer cells by preventing their development of a new blood supply: (2) suppressing development of kidney diseases due to cytokine induced proliferation of mesangial cells and/or glomerular epithelial cells that is useful for treating or preventing progression of diabetic glomerulosclerosis and other glomerulonephritis of various types and etiologies; (3) preventing joint destruction accompanying rheumatoid arthritis due to proliferation of synovial cells; (4) suppressing manifestations of psoriasis due to proliferation of keratinocytes and accumulation of inflammatory cells; (5) suppressing accelerated atherogenesis involved in restenosis of coronary vessels or other arterial vessels following angioplasty; (6) suppressing atherogenesis, coronary artery disease and other vasculopathies due to atherogenesis; and (7) suppressing tumor growth via paracrine or autocrine mediated responses to other cytokines such as PDGF, FGF EGF or VEGF that is useful for treating or preventing progression of tumors such as breast cancer stimulated through overexpression of her-2-neu receptor, wherein the inventive method comprises administering a compound that inhibits signal transduction.
The present invention is useful in developing methods that are used in the iterative drug design process. The process identifies potential agonists and antagonists to VEGFR-2 by de novo design of novel drug candidate molecules which bind to the VEGFR-2 receptor to improve their potency. The x-ray crystallographic coordinates disclosed herein, will allow generation of 3-dimensional models of the catalytic site and drug binding site of the VEGFR-2 protein.
De novo design primarily consists of the generation of molecules via the use of computer programs which build and link fragments or atoms into a site based upon steric and electrostatic complementarity, without reference to substrate analog structures. The drug design process begins after the structure of a target RTK is solved to at least a resolution of 2.8 xc3x85. Refinement of the structure to a resolution of 2.5 xc3x85 or better, with xe2x80x9cfixedxe2x80x9d water molecules in place provides more optimal conditions to undertake drug design.
It is another object of this invention to identify KIDs of proteins in the RTK family and develop deletions in said KIDs such that the proteins will be crystallizable and suitable for measurement by x-ray crystallographic means.
It is a further object of this invention to disclose a process whereby KID regions from a member of the RTK family of genes such as PDGF, EGF, VEGF and others are modified by deletion of amino acids from the KID regions so as to impart favorable physical characteristics of the resulting polypeptide product. Examples of such favorable physical characteristics are increased solubility, greater stability to temperature variations making the polypeptide suitable for analysis by nuclear magnetic resonance, high throughput screening, biochemical characterizations, x-ray crystallography, calorimetry and other diagnostic means.
It is yet another object of this invention to developing screening methods used in the drug design process of potential agonists and antagonists to proteins in the RTK family by de novo design of novel drug candidate molecules with potentially nanomolar potencies. The x-ray crystallographic coordinates disclosed based on the deletion mutated KIDs and various other deletions of said proteins in the RTK family, will allow generation of 3-dimensional models of the active binding site of the proteins in the RTK family.
In the following description, xe2x80x9cVEGFR2xcex9450xe2x80x9d corresponds to SEQ ID NO: 5, and xe2x80x9cPDGFRxcex1xe2x80x9d refers to SEQ ID No: 6.